IWC Annual Report 2003 - Institut für Wasserchemie und chemische ...

Dark chamber
with flow cell
CCD camera
14
Lens
Syringe pumps with sample, antibody
solutions and substrate and washing buffer
1.3 Bioanalytics II
1.3.1 Development of a Biosensor for the Rapid and Simultaneous Detection of
Antibiotics in Milk
Funding: Forschungskreis der Ernährungsindustrie, FEI
Cooperation: LMU Munich (Prof. Märtlbauer)
The presence of antibiotic residues in milk due to improper use can lead to allergic
reactions and the harm of the intestinal flora. There is also concern about increasing
bacterial resistance to antibiotics. In the dairy industry, contaminated milk can result
in the production problems of cultured products (yoghurt and cheese) due to the
inhibition of starter cultures causing significant economic losses.
Out
Sample loop
Chip with microarray
Flow cell
Laptop
In
For consumer protection, regulatory authorities have established
residue limits (MRL) for several antibiotics in bovine milk
(EC Regulation 2377/90). Microbial inhibition (e.g., agar diffusion)
assays are commonly used as screening tests. One disadvantage
of these methods is the duration of about three hours.
Usually the results are obtained, when the milk is already in
production. For this reason dairies use quick tests which can
detect the frequently used β-lactam antibiotics before the milk
is pumped out of the dairy van to avoid troubles with fermentation
steps in the milk processing. Other antibiotic groups than
the β-lactams can not be determined with these tests. Furthermore,
the dairy has to dispose the whole milk of a diary van
in case of a positive test result. To avoid the contamination of
the already collected milk a sensor would be very helpful, which
enables the rapid detection of all relevant antibiotics before the
milk is pumped into the diary van.
The basis for this sensor is the PASA system (Parallel Affinity Sensor Array) which
allows the transfer of an indirect ELISA onto a biochip. Analyte molecules are immobilized
as haptens in an array of spots on a biochip. The glass chip is silanized
to obtain reactive epoxy groups on the surface. Hapten protein conjugates can bind
covalently and by adsorption. The spots are created by a non-contact spotting system
with a piezo pump. The diameter of the spots is about 350 µm with a spot distance of
0.6 mm. The disposable chip is inserted in a flow cell of about 100 µL volume, where
all incubations and reactions are carried out automatically.
At present the simultaneous detection of the following analytes is possible in whole
milk (detection limits and MRLs in brackets): Penicillin G (3.3 µg/L, MRL: 4 µg/L),
cloxacillin (0.29 µg/L, MRL: 30 µg/L), cephapirin (0.12 µg/L, MRL: 60 µg/L), sulfadiazine
(3.49 µg/L, MRL: 100 µg/L), sulfamethazine (4.93 µg/L, MRL: 100 µg/L),
streptomycin (5.1 µg/L, MRL: 200 µg/L), gentamicin (12.1 µg/L, MRL: 100 µg/L),
neomycin (31.8 µg/L, MRL: 1500 µg/L), erythromycin (0.36 µg/L, MRL: 40 µg/L)
and tylosin (0.95 µg/L, MRL: 50 µg/L). Penicillin G could be detected at the maximum
residue limit (MRL), the detection limits for all other analytes were far below
the respective MRLs. The tests require no sample preparation and can be carried
out within 4 minutes 50 seconds. For the quantification of samples first a calibration
with 5 chips is performed. The test of samples each spiked with five analytes could be
demonstrated successfully. Different fat contents of the milk had no influence on the
results.
(B. Knecht)